WO2014090311A1

WO2014090311A1 - Iron oxide nanoparticles with a graphene coating

Info

Publication number: WO2014090311A1
Application number: PCT/EP2012/075399
Authority: WO
Inventors: Thomas Simmet; Berthold BUECHELE; Oleg LUNOV
Original assignee: Universitaet Ulm
Priority date: 2012-12-13
Filing date: 2012-12-13
Publication date: 2014-06-19

Abstract

The invention relates to a nanoparticle (1) with a core (2) and a graphene coating (3), wherein the core (2) contains iron oxide. Furthermore, a use of the nanoparticle (1) according to the invention in an imaging method, in a method for the localised induction of hyperthermia and in a method for the detection, analysis and/or separation of cells, protein (5) and/or nucleic acids (7) is described. Finally, a method for the production of the nanoparticle (1) comprising the co-precipitation of iron oxide and graphene is claimed.

Description

Iron oxide nanoparticles with a graphene coating

Background of the invention

The invention relates to a superparamagnetic nanoparticle with a core and a graphene coating. The invention further relates to the use of such a nanoparticle. Additionally, the invention comprises a method for the production of a nanoparticle.

Prior art

It is well known that superparamagnetic nanoparticles can be formed from ferromagnetic or ferrimagnetic materials. Such superparamagnetic nanoparticles can be used in medical imaging as well as compound separation and purification, for example, nucleic acid, protein and cell purification. In such applications, a high magnetisation (as measured in emu/g) is often desirable to increase imaging sensitivity and purification efficiency. While high magnetisations have been reached in nanoparticles containing cobalt, such nanoparticles are of limited use in biological systems due to the inherent toxicity of cobalt.

In the publication "TEMPO supported on magnetic C/Co nanoparticles: A highly active and recyclable organocatalyst", Chem. Eur. J. 2008, 14, 8262 - 8266, A. Schatz et al. report graphene coated nanobeads with a magnetic cobalt core, which were created on a large scale by reducing flame synthesis. TEMPO was grafted on the nanobeads using a "click"- chemistry protocol. The heterogeneous TEMPO-nanobeads can function as a highly active catalyst for the chemoselective oxidation of primary and secondary alcohols using bleach as terminal oxidant.

The patent application US 2008 / 0213189 A1 discloses nanocrystals comprising metals and metal alloys, which are formed by a process that results in a layer of graphite in direct contact with the metallic core. Preferred metals include iron, gold, cobalt, platinum, ruthenium and mixtures thereof, for example FeCo and AuFe. The nanocrystals may be used in vivo as MRI contrast agents, X-ray contrast agents, near IR heating agents, in drug delivery, protein separation or catalysis. The nanocrystals may be further functionalised with a hydrophilic coating, which improves in vivo stability. The nanocrystals are prepared by chemical vapour deposition and exhibit a high saturation magnetisation, high optical absorbance by the graphitic shell in the near-infrared and remarkable chemical stability. The saturation magnetisation of 7 nm FeCo graphite coated nanocrystals was 215 emu/g, close to bulk FeCo (235 emu/g).

The international patent application WO 2012 / 001579 A1 describes a method for forming iron oxide nanoparticles. The disclosed iron oxide nanoparticles are water-soluble and show superior performance in magnetic particle imaging and magnetic particle spectroscopy due to the high saturation magnetisation of 107 emu/g, which is reached in one embodiment.

Problem according to the invention

It is the underlying problem of the current invention to provide an improved nanoparticle and a process for the manufacture of such an improved nanoparticle. A further problem according to the invention is to provide a use for the nanoparticle. In particular, it is desirable for the nanoparticle to show a low toxicity and high magnetisation while being suitable for use in vivo as well as in vitro.

Solution according to the invention

The problem according to the invention is solved by a nanoparticle with a core and a graphene coating, wherein the core contains iron oxide. Furthermore, the problem is solved by the use of the nanoparticle according to the invention in an imaging method or the localised induction of hyperthermia. In addition to this, the use of the nanoparticle in a method for the detection, separation and/or isolation of cells, protein and/or nucleic acids serves to solve the problem according to the invention. Finally, the problem is also solved by a method for the production of the nanoparticle according to the invention that comprises the co-precipitation of iron oxide and graphene.

The nanoparticle according to the invention has a core containing iron oxide. That is to say, the core is either made up of pure iron oxide or it consists of a mixture of two or more materials, one of which is iron oxide. For example, the core material can be a mixture of iron oxide and other metal oxides. In another example, the core material is an alloy of several components, one of which is iron oxide. Iron oxide is any one or a mixture of two or more of the following compounds: FeO, Fe₂0₃ and Fe₃0₄. The nanoparticle according to the invention further comprises a graphene coating, wherein the surface of the core is at least partly covered with a graphene coating. The preferred nanoparticle has a substantially spherical shape. Preferably, the core and graphene coating each take substantially spherical shapes that are preferably in a substantially concentric arrangement. The invention, however, is not limited to spherical nanoparticles. Rather, the nanoparticles according to the invention can assume any shape, in particular, they can be rod-like or irregularly shaped. The nanoparticle according to the invention measures less than 500 nm across. Within the scope of this document, the diameter of the nanoparticle is defined as the equivalent spherical diameter, that is, the diameter of a sphere of equivalent volume.

The nanoparticle according to the invention can be used in an imaging method. An imaging method is any method suitable for the production of an image from a sample or test subject, such as an animal or a human being. Due to its achievable, high magnetisation, the nanoparticle according to the invention is especially suited for the use in magnetic resonance imaging and magnetic particle imaging.

The nanoparticle can be used in a method for the localised induction of hyperthermia.

Characteristically, nanoparticles injected into the bloodstream preferentially distribute to tumour sites as the tumour-adjacent vasculature often shows increased permeability.

Moreover, nanoparticles with surface structures aimed against specific tissues in the body can be injected into the vasculature, from where they specifically distribute to target sites. The subsequent application of an external, alternating magnetic field can heat the nanoparticles, causing the target tissues to be damaged. This approach is especially successful in cancer therapy as many cancer tissues show less heat tolerance than the surrounding healthy tissue.

The nanoparticle according to the invention can also be used in the detection, analysis separation and/or isolation of cells, protein and/or nucleic acids. In order for target cells, protein and/or nucleic acids to be separated from a solution, a nanoparticle conjugated to a protein or nucleic acid with a high affinity to the target can be employed. In particular, antibodies against target antigens are suitable for such applications. The protein conjugated nanoparticle can be attached to a target protein or a surface protein of a target cell. If the nanoparticle is conjugated to a nucleic acid, it can bind to complimentary nucleic acids. After the nanoparticles have bound to their targets, a magnetic field can be applied that separates the nanoparticles along with their bound target proteins, cells and/or nucleic acids from the solution. In order to detect a protein, cell or nucleic acid in solution, nanoparticles can first be attached to a potential target in the aforementioned procedure. In a second step, a secondary reagent, such as a fluorescent labelled antibody or nucleic acid specific for the target can be added. The nanoparticles can be collected in a magnetic field and tested for the presence of the secondary reagent, for example, by excitation of fluorescence, much like in a sandwich ELISA assay, the conjugated nanoparticles being equivalent to the plate coated in capture antibody and the secondary reagent being equivalent to the detecting antibody. If a target is to be analysed on a surface, nanoparticles conjugated with structures having a high affinity to the target can be brought into contact with a surface. After a washing step, the concentration of the target on the surface can be estimated using a magnetometer or a magnetic resonance system.

The invention further relates to a method for the production of the nanoparticle according to the invention comprising the co-precipitation of iron oxide and graphene. The nanoparticle can, for example, be produced by flame spray synthesis, high temperature decomposition of organic precursors, formation in water-in-oil microemulsions and co-precipitation, amongst others. Co-precipitation can be achieved by mixing FeCI₂, FeCI₃ and graphene.

Subsequently, when ammonia solution is added, precipitation of iron oxide nanoparticles coated with graphene is achieved.

Advantageously, the composition of the nanoparticle according to the invention allows for a high magnetisation, previously not attained in a non-toxic biocompatible nanoparticle.

Particularly for the use in biological systems, the nanoparticle makes it possible to increase temporal and spatial resolution as wells as sensitivity in imaging methods. The nanoparticle can be used in medical applications in vivo in humans and animals as well as in vitro laboratory tests. Furthermore, the invention discloses a simple method for the production of the nanoparticle according to the invention.

Specific embodiments according to the invention

The diameter of the core, the graphene coating including the core and/or the entire nanoparticle according to the invention is preferably >1 nm, more preferably >2 nm, more preferably >5 nm, more preferably >10 nm and most preferably >15 nm. At the same time, it is preferred that the core, the graphene coating including the core and/or the entire nanoparticle according to the invention is <400 nm, more preferably <250 nm, more preferably <150 nm, more preferably <100 nm, more preferably <80 nm, more preferably <60 nm, more preferably <40 nm, more preferably <25 nm and most preferably <20 nm in diameter. The core of the nanoparticle according to the inventions contains iron oxide.

Preferably, the core is made up of a mixture of materials containing >10%, more preferably >25%, more preferably >50%, more preferably >75%, more preferably >90%, more preferably >95% and most preferably >99% (weight/weight) iron oxide. It is preferred that the core of the nanoparticle consists entirely of iron oxide, of which preferably >99%

(weight/weight) is Fe₃0₄, most preferably, the entire core consists of Fe₃0₄ or a mixture of Fe₃0₄ and FeO or Fe₂0₃. Iron oxide is any one or a mixture of two or more of the following compounds: FeO, Fe₂0₃ and Fe₃0₄.

According to the invention, it is preferred that the nanoparticle is superparamagnetic. The preferred nanoparticle according to the invention is small enough that superparamagnetism can occur. Advantageously, superparamagnetic iron oxide particles have a large, positive magnetic susceptibility, as the entire particle aligns with and strengthens the applied magnetic field leading to a local disturbance. When magnetic resonance imaging is performed, the local disturbance can lead to a rapid dephasing of surrounding protons, generating a detectable change in the magnetic resonance signal. Thus, superparamagnetic iron oxide particles may easily be detected on magnetic resonance imaging. Furthermore, superparamagnetic iron oxide particles can be sufficiently small for the Brownian motion to demagnetise the particles once an applied field is taken away. Thereby, the aggregation of the superparamagnetic iron oxide particles in solution due to magnetic attraction is prevented.

It is preferred that the nanoparticle according to the invention shows a magnetisation of >120 emu/g. Preferably, the magnetisation is >150, more preferably > 180, more preferably > 190, more preferably >200 and most preferably >205 emu/g. A large magnetisation may allow for the easy detection in magnetic resonance imaging and magnetic particle imaging.

Furthermore, a large magnetisation can considerably facilitate the use of the nanoparticles in cell, protein and nucleic acid separation and analysis.

The preferred nanoparticle according to the invention has a graphene coating that covers at least 50% of the surface of the core with between 1 and 5 layers of graphene. Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the core is covered with between 1 and 5 layers of graphene. Preferably, no part of the surface of the core is covered with more than 20, more preferably more than 10 and most preferably more than 5 layers of graphene. Experiments have shown that a thin graphene coating of between 1 and 5 layers is sufficient to protect the iron oxide particles from decay and degradation. Furthermore, the iron oxide nanoparticle according to the invention covered in between 1 and 5 layers of graphene shows a high magnetisation. The preferred nanoparticle according to the invention has a graphene coating that covers at least 50% of the surface of the core with exactly 3 layers of graphene. Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the core is covered with exactly 3 layers of graphene. The inventors have found that a nanoparticle with 3 layers of graphene in its coating is well protected against degradation and shows a high magnetisation while being relatively small. The preferred nanoparticle according to the invention has a graphene coating that covers at least 50% of the surface of the core. Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the core is covered with the graphene coating. By completely covering the nanoparticle with the graphene coating, an optimum protection against degradation is achieved. In addition to this, a good approximation to a spherical shape can be yielded.

The preferred nanoparticle according to the invention has a graphene coating that is covered entirely in one additional layer or several additional layers. Preferably, the nanoparticle has 2, 3 or 4 additional layers. Preferably, the additional layer or the additional layers cover at least 50%, more preferably at least 75%, more preferably at least 90%, more preferably at least 95% and most preferably at least 99% of the surface of the core and/or of the graphene coating. An additional layer can facilitate the functionalisation of the nanoparticle.

Furthermore, an additional layer can be used to render the nanoparticle hydrophobic or hydrophilic, to make the particle inert and resistant to solvents or the biological environment. In a preferred nanoparticle according to the invention, at least one layer of the additional layers consists of a polymer. Using a polymer, the nanoparticle according to the invention can be made into almost any shape with almost any surface structure. Furthermore, by using a suitable polymer, an inert nanoparticle resistant to decay can be produced.

The nanoparticle according to the invention is preferably functionalised with a protein or a nucleic acid. By functionalising the nanoparticle with a protein, such as an antibody, other proteins or cells can be labelled with the nanoparticles. Nucleic acids can be labelled with nanoparticles conjugated to complementary nucleic acids. By application of a magnetic field, cells, proteins and nucleic acids can then be separated or analysed. The preferred nanoparticle according to the invention is functionalised with a drug. Drugs according to the invention include low molecular compounds such as vitamins, for example, folic acid. The drug can - depending on the individual structure of the drug - be conjugated to the nanoparticle or absorbed at its surface. The drug can either be attached to the graphene layer or to one of the additional layers. In principle, the nanoparticle according to the invention can be functionalised with any drug. Drugs that are targeted against localised disorders such as infections or neoplastic lesions are especially suited for the

functionalisation on nanoparticles. In one embodiment, the drugs are stored in vesicles on the surface of the nanoparticle. The drug conjugated nanoparticles can work as a

theranostic, a portmanteau of the words therapeutic and diagnostic. In one embodiment, the nanoparticles according to the invention can be injected into the human body, for example via an intravenous route. They then distribute themselves from the injection site to the target tissue. The distribution can be controlled by a further functionalisation of the nanoparticles, for example, an antibody directed against a cancer antigen or a protein allowing for endocytosis of the nanoparticle in a particular, targeted species of cells. In the diagnostic part of the theranostic, the nanoparticles are then monitored by magnetic resonance imaging or magnetic particle imaging to find out, whether they have distributed to the target site with sufficient precision. Furthermore, the same imaging techniques can be used to ascertain that the drug functionalised nanoparticles have not spread to vital tissues not to be treated with the drug. In a second step, the drug is released at the target site. This can either occur simply through the passage of time as the drug slowly dissociates itself from the

nanoparticle. Alternatively, an external alternating magnetic field can be applied to the nanoparticles, which causes the nanoparticles to be heated and thereby release the drug. Such alternating magnetic fields can be created using specialised machinery or magnetic resonance coils adapted to the purpose. Thus, in one embodiment, the nanoparticles according to the invention represent a bona fide theranostic in that they can be used to both find pathologies as well as to cure them.

Additionally, the nanoparticle according to the invention can be used in a method for the analysis of protein-protein and/or protein-nucleic acid interactions. In medical research, it is often desirable to find protein-protein interaction partners, in basic research as well as in drug design. To this end, the nanoparticle can be conjugated with a first protein, which is a putative interaction partner of a second protein in a solution. The nanoparticle conjugated to the first protein can be added to that solution and subsequently, by using centrifugation or the application of a magnetic field, can be separated from the solution again. Using an antibody specific for the second protein, it can be detected, whether the first protein on the surface of the nanoparticle has bound to the second protein. Similarly, this method can be applied using a nanoparticle that has been conjugated with an antibody specific for the first protein.

Furthermore, it can be of interest at which exact position certain proteins interact with nucleic acids. In particular, the localisation of transcription factor binding sites on DNA is a common object of scientific inquiry. If an antibody to a transcription factor or the transcription factor itself is conjugated to the nanoparticle, the binding site of the transcription factor can be found by adding the said conjugated nanoparticle to a pool of genomic DNA, denaturing the protein to make the DNA-protein connection irreversible, subsequently removing the nanoparticle bound to DNA using a magnetic field and applying a DNA identification technology such as sequencing to characterise the DNA bound to the transcription factor. The high achievable magnetisation of the nanoparticle can increase yield and/or sensitivity in the aforementioned applications.

In one embodiment of the invention, the nanoparticles are used in an imaging method, which is magnetic resonance imaging or magnetic particle imaging. The nanoparticles according to the invention can be produced to have a large magnetisation. Such nanoparticles, when introduced into the magnetic field of an magnetic resonance scanner cause a large, localised disturbance in the magnetic field, which in turn causes surrounding protons to dephase rapidly, leading to a loss of T2 signal. As the disturbance induced by a single nanoparticle according to the invention is usually greater than with other biocompatible nanoparticles known in the art, a higher sensitivity in magnetic resonance applications can be achieved. That is, in order to produce a detectable change in a magnetic resonance image, fewer nanoparticles according to the invention are needed than would be necessary when using nanoparticles from known in the art. Advantageously, the nanoparticles according to the invention can also be used in magnetic particle imaging. Magnetic particle imaging can measure the localisation of magnetic material in a given volume. To achieve high temporal as well as spatial resolution in magnetic particle imaging, it is desirable to utilise a nanoparticle with a very large magnetisation, which can be supplied by the invention.

Preferentially, the method for the production of the nanoparticle comprises the co- precipitation of iron oxide and graphene using FeCI₂, FeCI₃as precursors. Experiments have shown that the precipitation of FeCI₂, FeCI₃ and graphene in an aqueous solution by the addition of ammonia solution is a very efficient method to produce the nanoparticle according to the invention. It is preferred that the method for the production of the nanoparticle comprises the conjugation of the nanoparticle to a protein, nucleic acid and/or drug. By conjugation of the nanoparticle to a protein and/or nanoparticle, specific structures can be targeted. In particular, the nanoparticle can be made to bind to specific tissues in the human body for use in, for example, imaging or drug delivery of a conjugated drug. Proteins, nucleic acids and drugs can be conjugated to the nanoparticles according to a number of procedures known in the art, such as layer-by-layer with 1 -ethyl-3-[3-dimethylaminopropyl]carbodiimide or using 1 - ethyl-3-[3-dimethylaminopropyl]carbodiimide with polyethyleneimine.

The invention provides an improved nanoparticle, several uses for the nanoparticle and a method to produce such a particle. In particular, the invention allows for the production of a nanoparticle with a low toxicity and a high magnetisation.

Brief description of the figures

The invention is explained in detail in the following figures. The figures show:

Fig. 1 : the nanoparticle according to the invention in a schematic representation;

Fig. 2a: an electron micrograph of iron oxide nanoparticles;

Fig. 2b: an electron micrograph of iron oxide nanoparticles with a graphene coating; Fig. 2c: a diagram depicting size measurements obtained by dynamic light scattering and

Fig. 2d: a schematic representation of the nanoparticle according to the invention.

Description of specific embodiments of the invention

Fig. 1 depicts a spherical nanoparticle 1 with an iron oxide core 2 surrounded by several layers of graphene in a graphene coating 3. By applying the graphene coating 3, a previously unattainable magnetisation of 215 emu/g can be achieved. Furthermore, the graphene coating 3 serves to protect the iron oxide core 2 of the nanoparticle 1 against disintegration and decay. Optionally, an additional layer 4 consisting of a polymer is applied on top of the graphene coating 3. The additional layer 4 further shields the nanoparticle 1 from its environment. The nanoparticle 1 can be functionalised by attaching proteins 5, such as antibodies 6, to the additional layer 4. Alternatively or in addition to proteins 5, the nanoparticles 1 can also be conjugated to nucleic acids 7 and drugs 8, particularly antibiotic or antineoplastic drugs.

Fig. 2a shows a scanning electron microscopic image of iron oxide nanoparticles 1 without a surface coating. The uncoated nanoparticles 1 have a mean diameter of 12 nm. Fig. 2b displays the nanoparticles 1 according to the invention covered in a graphene coating 3 in a scanning electron micrograph. The graphene coating 3 has added to the size of the nanoparticles 1 , which now measure 17 nm across on average. Fig 2c contains a diagram of the result of a dynamic light scattering experiment. The nanoparticle diameter is shown on the x-axis while the y-axis displays light intensity as a percentage value. The dashed curve on the left corresponds to the nanoparticles 1 without coating as displayed in fig. 2a. The nanoparticles 1 with the graphene coating 3 from fig. 2b correspond to the continuous curve slightly to the right. From this diagram it is clear to see that the graphene coating 1 has added to the mean diameter of the iron oxide nanoparticles 1. Fig. 2d is a schematic representation of an iron oxide nanoparticle 1 according to the invention covered in a graphene coating 3 with its characteristic hexagonal, honeycomb lattice composition.

The nanoparticles 1 according to the invention can be used in a variety of applications in vivo and in vitro. In an embodiment, the nanoparticles 1 can be functionalised with proteins 5, in particular with antibodies 6, and can be utilised, for example, in protein isolation, kinase assays, detection of protein-protein interactions, protein-nucleic acid interactions, such as chromatin immunoprecipitation and to separate cells from suspensions, such as blood. Furthermore, the nanoparticles 1 can be employed in vivo in imaging methods, such as magnetic resonance imaging and magnetic particle imaging. The high magnetisation that can be achieved in the nanoparticles 1 according to the invention can improve sensitivity as well as temporal and spatial resolution in these imaging modalities.

Protocols for the production of iron oxide nanoparticles with a graphene coating

The following protocols demonstrate one method for the production of the nanoparticles according to the invention. By adhering to the protocols below, nanoparticles covered in three graphene layers and with a high magnetisation of 215 emu/g can be produced. The invention, however, is not limited to the method outlined in these protocols. Rather, other production methods are equally feasible. The production of graphene - as described below - comprises the steps of synthesis of graphene oxide and the subsequent reduction of graphene oxide to graphene. In a third step, superparamagnetic iron oxide particles with a graphene coating are produced by co-precipitation.

Synthesis of graphene oxide

60 mg graphite powder (<20 μιη) and 45 mg sodium nitrate are placed in a 100 ml round- bottom flask, the flask is closed with a drying tube. 4.5 ml of sulphuric acid is added during 10 minutes while stirring and cooling. Further stirring is performed on ice for 2 h. After that, stirring is carried out for 6 days at room temperature. Subsequently, 7 ml 5% sulphuric acid in water (w/w) is added. The mixture is stirred for 2 h at 98 °C. Then, the temperature is lowered to 60 °C. 0.2 ml hydrogen peroxide is added, stirring is performed for 1 h. The solution is centrifuged at 16,060g for 2 minutes. The sediment is homogenised in 1.7 ml 3% sulphuric acid, 0.5% hydrogen peroxide (w/w). Homogenisation and centrifugation is repeated 15 times. The sediment is then spun in a centrifuge at 16,060g and washed in 1 ,7 ml 3% sulphuric acid for a total of 3 times. Next, the sediment is suspended in water and homogenised in an ultrasonic bath. After that, the suspension is spun at 16,060g for 15 minutes and the sediment is dried at 10^~1 torr. The achievable yield is approximately 50 mg graphene oxide.

Synthesis of grapheme from graphene oxide by chemical reduction with hydrazine

10 mg graphene oxide from the previous step is suspended in 10 ml ultra-pure water and placed in an ultrasonic bath for 1 minute. 112 μΙ 32% ammonia solution is added within 5 minutes. 18 μΙ 62% hydrazine solution is added within 5 minutes. The suspension is stirred for 1 h at 90 °C under reflux. The entire preparation is then transferred into Falcon tubes and spun in a centrifuge for 3 minutes at 4,600g. The supernatant is transferred into 20 ml round- bottom flasks und concentrated under vacuum at 80 °C in a rotary evaporator. The residue is dried at 10^~1 torr. The achievable yield is approximately 7 mg graphene.

Production of superparamagnetic iron oxide nanoparticles with a graphene coating

25 mg FeCI₂ x 4 H₂0 (final concentration 5 mM) and 68 mg FeCI₃x 6 H₂0 (final concentration 10 mM) are dissolved in 25 ml ultra-pure water saturated with nitrogen. To the resulting solution, 1 ml graphene solution (5 mg/ 10 ml 1% (v/v) ammonia solution, pH 8.5) is added under nitrogen saturation. 5.8 ml 32% ammonia solution is added drop by drop within 30 minutes while thoroughly mixing with nitrogen. The mixture is kept at 70 °C for 30 minutes, during which time the nanoparticles precipitate. The nanoparticles are separated using an external magnet. The supernatant is decanted, the nanoparticles are washed twice in 10 ml water and twice in methanol. After that, the nanoparticles are homogenised in 10 ml toluene in an ultrasonic bath (2 mg Fe per ml solvent). For stabilisation and storage, the

nanoparticles are kept in a nitrogen atmosphere at 4 °C.

Reference numbers

1. Nanoparticle

2. Core

3. Graphene coating

4. Additional layer

5. Protein

6. Antibody

7. Nucleic acid

8. Drug

Claims

1. Nanoparticle (1) with a core (2) and a graphene coating (3), characterised in that the core (2) contains iron oxide.

2. Nanoparticle (1) according to claim 1 , characterised in that the nanoparticle (1 ) is superparamagnetic.

3. Nanoparticle (1) according to claim 1 , characterised in that the nanoparticle (1) shows a magnetisation >120 emu/g.

4. Nanoparticle (1) according to any of the preceding claims, characterised in that the graphene coating (3) covers at least 50% of the surface of the core (2) with between 1 and 5 layers of graphene.

5. Nanoparticle (1) according to any of the preceding claims, characterised in that the graphene coating (3) covers at least 50% of the surface of the core (2) with exactly 3 layers of graphene.

6. Nanoparticle (1) according to any of the preceding claims, characterised in that the graphene coating (3) covers at least 50% of the surface of the core (2).

7. Nanoparticle (1) according to any of the preceding claims, characterised in that the graphene coating (3) is covered entirely in one additional layer (4) or several additional layers (4).

8. Nanoparticle (1) according to claim 7, characterised in that at least one layer of the additional layers (4) consists of a polymer.

9. Nanoparticle (1) according to any of the preceding claims, characterised in that the nanoparticle (1) is functionalised with a protein (5) or nucleic acid (7).

10. Nanoparticle (1) according to any of the preceding claims, characterised in that the nanoparticle (1) is functionalised with a drug (8).

1 1. Use of the nanoparticle (1) according to any of the preceding claims in an imaging method.

12. Use according to claim 11 , characterised in that the imaging method is magnetic resonance imaging or magnetic particle imaging.

13. Use of the nanoparticle (1) according to one of the claims 1 to 10 in a method for the localised induction of hyperthermia.

14. Use of the nanoparticle (1) according to one of the claims 1 to 10 in a method for the detection, analysis and/or separation of cells, protein (5) and/or nucleic acids (7).

15. Method for the production of the nanoparticle (1) according to any one of the claims 1 to 10, characterised in that it comprises the co-precipitation of iron oxide and graphene.

16. Method according to claim 15, characterised in that the method comprises the conjugation of the nanoparticle (1) to a protein (5), nucleic acid (7) and/or drug.